Staphylococcus aureus is a versatile human pathogen that is responsible for both healthcare-associated infections and, increasingly, community acquired disease. S. aureus causes a wide range of diseases, from minor skin and soft tissue infections to more serious disorders including pneumonia, toxic shock, osteomyelitis and endocarditis. It is commonly found associated with infection of indwelling medical devices and surgical wounds. The emergence of aggressive strains resistant to multiple antibiotics, in combination with widespread horizontal gene transfer, poses a significant public health hazard. The ability of S. aureus to adapt rapidly to antibiotic pressure underscores the need for continued development of new targets for antimicrobial therapy. Recent work in our laboratory has led to the discovery that one of the proteins in the large subunit of the ribosome is subject t a specific N-terminal cleavage in S. aureus and other Gram positive bacteria. Several lines of evidence are consistent with the hypothesis that this cleavage event is essential. Bioinformatic and biochemical studies have led to the identification of the protease responsible for this cleavage. This proposal includes a combination of genetic and biochemical approaches to confirm the requirement for cleavage and the characterization of the protease. We will develop a cleavage assay and identify key amino acid residues in the protease and in the ribosomal protein that are necessary for this targeted cleavage event. These studies will elucidate the role of this novel ribosomal protein processing event and set the stage for subsequent efforts aimed at the development of a new class of antibiotics that will inhibit this cleavage reaction.